Atypical Cystic Fibrosis

Question 1

What are the two categories of cystic fibrosis?

Answer 1

Classical

Atypical

Question 2

Describe atypical CF

Answer 2

Mild symptoms
Majority have 1 or no CFTR mutations
Carriers have no symptoms

Question 3

What is the hypotheis for ENaC mutations in atypical CF?

Answer 3

Gain of function mutation
Enhanced Na+ absorption causes a depletion of the PCL
This causes CF like symptoms as cilia are bent

Question 4

What was found when atypical CF patients were screened for ENaC mutations?

Answer 4

alpha = 11 mutations found
beta = 7 mutations found
gamma = 8 mutations found
Another study also showed similar findings and some mutations were seen again

Question 5

What was seen when different ENaC mutations were looked at for their amiloride sensitive current?

Answer 5

Some mutations showed a gain of function as ENaC currents were greater
However, some actually were loss of function and some did not have an effect

Question 6

Describe work which showed that in atypical CF patients their ENaC channels had gain of function

Answer 6

Nasal potential was measured
Normal patients - Vte was measured at -15mV
Low Cl- solution = shift to -16mV = functional CFTR
Add amiloride = shift of potential by approx 6mv =
functional ENaC
Classic CF - Vte is hyperpolarised at - 31mV
Low Cl- = shift of 0.08mV = no CFTR open
Add amiloride = shift of 19.4mV = ENaC function is
enhanced as CFTR is not there to inhibit ENaC
CF Carrier - Vte start potentialis approx -19mV
Low Cl- solution = shift to -20mV = normal CFTR
function
Add amiloride = shift of 18mV so ENaC is GOF
So we have excess Na+ reabsorption = PCL height depletion

Question 7

Write a summary for Atypical CF

Answer 7

Some GOF mutations in ENaC were found in atypical CF
These explain the CF like symptoms in patients
However, some LOF mutations have also been found
It is not known how these necessarily cause symptoms

Question 8

Describe the alpha W493R mutation

Answer 8

Found on the extracellular loop of the matrix
2010 study confirmed this was GOF in ENaC
It was overexpressed in cells on the two different ENaC backgrounds
Amiloride was used to measure function
ENaC currents in the mutant give a bigger increase in current when amiloride is washed out
Mutation enhances the current

Question 9

What is Na+ feedback inhibition?

Answer 9

ENaC channels are endocytosed from the apical membrane
When it opens Na+ floods in so there is a transient increase
This transient increase promotes removal of ENaC from the membrane
If this endocytosis is not happening then we see larger currents which stay higher for longer

Question 10

What did they see in terms of Na+ feedback inhibition in the alpha W493R mutant

Answer 10

The starting expression level was not different
In the high Na+ we should see lower currents when the ENaC is being endocytosed
In the mutant the initial currents were higher but there was still inhibition of ENaC
If we do a ratio the percentage drop in the WT and the mutant is similar
So the mutant Na+ feedback inhibition is normal

Question 11

Describe the cleavage of ENaC

Answer 11

Cleaved ENaC shifts to a higher Po - it is cleaved by proteases
Uncleaved = near silent, Low Po and smaller ENaC currents
Cleaved = very active, High Po and larger ENaC current

I = N.Po.g (Vm-Ei)

Question 12

What is chymotrypsin?

Answer 12

A protease which cleaves ENaC

Question 13

What is the response of ENaC to chymotypsin in WT and the mutant?

Answer 13

When added to the WT it increases current as ENaC Po is increased due to cleavage
However when it is added to the mutant the response is lost as there is no increase in current in the mutant
Does this mean the channel is already cleaved?
No - the channels just have a higher Po

Question 14

How was it shown that the mutant channels were not already and just had a higher Po?

Answer 14

The WT channels had a flickery opening in single channel recordings
However, the mutant had sustained opening events
Adding chymotrypsin to the WT increases the number of channels open but in the mutant the channels are already open so there is no effect
So this is not due to cleavage

Question 15

What is sodium self inhibition?

Answer 15

ENaC channels means that the amount of Na+ going on to the cell can regulate the Po of the ENaC

Question 16

How is sodium self inhibition different between WT and the mutant ENaC channel?

Answer 16

In the WT adding/ increasing extracellular Na+ causes an initial increase in current but this decreases slightly due to the Na+ self inhibition
However in the mutant channel this appears to be lost
So the small decrease in current has gone
This is enough to mean that Na+ channels in patients have problems

Question 17

What did researchers want to find out about the BV348M mutation?

Answer 17

They wanted to see if this GOF mutation had an increased Po

Question 18

How did they see what the BV348M was doing?

Answer 18

They used MTSET - this is a sulfhydryl agent which binds to cysteine to stabilise the channel in the open state i.e. Po = 1
Measuring current before and after MTSET gives apparent Po
For MTSET to work they had to use a mutant BS520C to introduce a cysteine
WT Po = 0.24
Mutant Po = 0.33
So mutant channels have an increase in Po = more Na+ reabsorption

Question 19

What was seen at the single channel level between the WT and the mutant?

Answer 19

WT opening was flickery

Mutants have sustained opening events

Question 20

What happens in mice who overexpress the B subunit SCNN1b?

Answer 20

B subunit expression is the rate limiting step for ENaC function
Overexpressing the B subunit caused a decrease in PCL height and so the cilia are bent over
This is seen in both the bronchus and the trachea
In these mice mucus clearance was also significantly reduced and was associated with mucous plaques and plugs
This was associated with significant postnatal mortality

Question 21

How was mucous clearance affected in mice overexpressing the B subunit?

Answer 21

WT mice cleared the bacteria

However the mutant animals could not clear the mucous and therefore did not clear the mucous from the lungs